Coming dissertations at MedFak
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A Comprehensive Analysis of Endothelial Cell Subpopulations in Health and Disease Using Single-Cell Transcriptomics
Link: http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-539669
The vascular system consists of blood vessels, lymphatic vessels, and hybrid vessels, each with unique functions defined by specialized endothelial cells (ECs). These ECs show remarkable heterogeneity, adapting to organ- and disease-specific conditions. In this work, we used single-cell RNA sequencing (scRNA-seq) to define the transcriptome of normal blood endothelial cells (BEC), lymphatic endothelial cells (LEC) and hybrid vessel ECs from selected mouse and human tissue. Through this detailed analysis we identified a new population of sinusoidal ECs with a unique hybrid vessel identity in the penile vasculature described in paper I. In addition, in paper II we uncovered a previously undescribed subtype of dermal capillary LECs in mouse skin, characterized by their expression of immune regulatory genes.
By utilizing scRNA-seq and genetic mouse models, we explored the molecular and cellular mechanisms underlying oncogenic PI3K-driven lymphatic and venous malformations (LM and VM, respectively) and showed in paper II and paper III that distinct subpopulations of LECs and BECs respond differently to the PI3K gain-of-function mutation Pik3caH1047R. Specifically, LECs respond by expanding the newly identified immune-interacting capillary subpopulation, which promotes the recruitment of pro-lymphangiogenic myeloid cells. In the blood vasculature, venous-like capillaries and post-capillary venules respond to PI3K activation through selective clonal expansion, leading to the formation of VMs. Furthermore, we identified a venous-specific regulatory feedback loop in paper III, involving the inhibition of the transcription factor FOXO1 and the activation of the Angiopoietin-TIE2 signalling pathway. This feedback loop promotes VM growth and represents a promising therapeutic target for developing effective treatments for VM patients.
Overall, the work presented in papers I-III significantly advanced our understanding of EC heterogeneity and function in normal tissues and vascular diseases, potentially paving the way for the development of new pharmacological targets.
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Scaffold Protein-based Theranostics of HER2-overexpressing Ovarian Cancer: Imaging-guided Therapy
Link: http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-539484
This thesis is based on five original articles aiming to develop HER2-targeting affibody-albumin-binding-domain (ABD)-drug conjugates (AffiDCs) for the treatment of ovarian cancer. The research focused on several aspects of molecular design, including the number of HER2-binding domains (ZHER2), the number and position of functional domains targeting HER2 or albumin, the number of conjugated drug molecules, the composition of linkers between domains, and the drug composition. The cytotoxic payloads DM1, MMAE or MMAF were conjugated via a maleimidocaproyl (mc) linker. All affibody-based constructs were radiolabeled with technetium-99m to quantitatively assess their properties in vitro and in vivo. The selected conjugates were evaluated in therapeutic studies using the HER2-overexpressing SKOV3 ovarian cancer xenograft model in BALB/c nu/nu mice.
In Paper I, the influence of HER2-binding valency on targeting properties, internalization, cytotoxicity, and drug delivery was evaluated. The ZHER2-ABD-E3-DM1 conjugate showed highly potent anti-tumor activity in vivo. Imaging using SPECT/CT visualized the HER2 expression during the treatment. In Paper II, the influence of the number and position of functional domains on cancer cell proliferation was investigated. While dimeric anti-HER2 affibody molecules stimulated the proliferation of cancer cells in vitro and promoted tumor growth in vivo, the additional stimulation of proliferation did not improve the therapeutic effect of DM1. ZHER2-ABD was selected as the most optimal format for targeted delivery of DM1. In Paper III, the influence of the number of conjugated drug molecules on biodistribution and tumor-targeted drug delivery was investigated. Increasing the DM1 number from one to three increased the amount of drug delivered to tumors; however, it also raised the risk of normal organ toxicity. In Paper IV, the influence of linker composition between the affibody domain and ABD on biodistribution was evaluated. Introducing additional serine in the inter-domain linker decreased the hepatic uptake. However, the linker length should be optimal to preserve cytotoxicity and high-affinity binding to the target. In Paper V, the impact of the drug composition on binding properties, cytotoxicity, and biodistribution was examined. ZHER2-ABD-MMAF showed higher efficacy than ZHER2-ABD-DM1 for HER2-targeted therapy in vivo.
In conclusion, modification of molecular design impacts functional properties, biodistribution, and tumor targeting of AffiDCs. Careful optimization of molecular design allowed for selecting several candidates with high anti-tumor efficacy and favorable toxicity profiles in mice.
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On the physiology of psychiatric brain stimulation
Link: http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-539059
This thesis examines physiological aspects of two psychiatric brain stimulation treatments: electroconvulsive therapy (ECT) and repetitive transcranial magnetic stimulation (rTMS).
ECT’s therapeutic effect depends on epileptic seizures that activate subcortical regions, and involves increased gamma-aminobutyric acid (GABA) inhibition. Clinical ECT seizure evaluation parameters show varying correlation to clinical outcome and uncertain correlation to subcortical activation. Serum prolactin and cortisol increase postictally, and postictal prolactin reflects subcortical seizure activation. The GABA-agonistic hormone allopregnanolone affects psychiatric illness and increases postictally in epilepsy, but is unexplored after ECT seizures.
RTMS’s therapeutic effect depends on altering cortical excitability which affects synapse plasticity, subsequently modulating functional neural network activity. Motor cortex excitability measured with TMS paired with electromyography (TMS-EMG), which includes GABA modulated indices, may be altered in psychiatric illness. Prefrontal rTMS affects motor cortex excitability, and TMS-EMG may predict rTMS treatment outcome, but the rTMS protocol intermittent theta burst stimulation (iTBS) is less explored.
Study I analysed correlations between clinical ECT seizure evaluation parameters and prolactin and cortisol serum levels. Ictal tachycardia correlated with postictal prolactin, but not cortisol, suggesting that ictal tachycardia reflects subcortical seizure engagement.
Study II investigated progesterone and allopregnanolone serum levels after an ECT seizure, finding no postictal increase or correlation to seizure evaluation parameters. Postictal progesterone increased in men in a subgroup analysis.
Study III examined GABAA receptor availability measured with positron emission tomography and TMS-EMG indices in participants with depression before and after iTBS. Baseline comparisons showed no difference from healthy. After iTBS, changes in GABAA receptor availability and TMS-EMG motor threshold correlated negatively, indicating GABAA mediated regulation of motor cortex signalling.
Study IV investigated whether iTBS affects TMS-EMG and whether baseline TMS-EMG predicts iTBS outcome on negative symptoms in schizophrenia and depression, with negative results. Compared to participants with depression and healthy, participants with schizophrenia had more GABAB receptor dependent inhibition, which also correlated with antipsychotic medication dose.
These results contribute to the understanding of ECT and rTMS physiology, facilitating future developments for improved efficacy and reduced side effects.